Cells interact with structurally and biochemically distinct types of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What unique mechanisms do mammalian cells use to migrate through three-dimensional (3D) extracellular matrix environments rather than on flat culture surfaces? 2. What distinct signal transduction mechanisms control cell behavior in different 3D microenvironments? We are exploring whether classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. We discovered a unique mode of 3D migration using high-resolution live-cell imaging to visualize intracellular signaling using different in vitro models of 3D extracellular matrix. Primary dermal fibroblasts migrating in dermal tissue explants and cell-derived matrix were found to use blunt, cylindrical protrusions termed lobopodia, named after an intracellular pressure-driven protrusion used by migrating amoeba. In contrast, cells migrating in 3D collagen gels exhibited lamellipodia-based migration similar to 2D cell culture, with small, fan-shaped protrusions enriched in F-actin at the leading edge. We have been comparing the localization of cytoskeletal and signaling systems in primary human fibroblasts migrating in different 3D matrix environments. Our studies recently demonstrated that even though active Rac1, Cdc42, and PIP3 are polarized towards the leading edge during lamellipodia-based migration in 3D collagen, during lobopodia-based migration, this polarization of Cdc42, Rac1, and PIP3 signaling is lost. Instead, signaling is concentrated in focal clusters behind leading protrusions, along the sides, and at the rear of the cell. Reducing actomyosin contractility switches the cells to lamellipodia-based 3D migration. In order to explore how a 3D extracellular matrix regulates mechanotransduction to control the mode of leading-edge protrusion, we are investigating the function of the intracellular cytoskeletal machinery during lamellipodia- and lobopodia-based migration. These studies are comparing the localization and function of fibrillar systems, including cytoskeletal systems based on myosin II and intermediate filaments. We are currently focusing particularly intensely on the potential role of intracellular pressure in lobopodial cell migration. Plasticity of cell migration in response to interaction with different extracellular matrix molecules is known to be necessary for efficient tissue development and wound repair, and it is often deregulated in cancer. Differential activation of the Rho family GTPases, Cdc42, Rac1, and RhoA, has been implicated in governing the distinct morphological and migratory phenotypes resulting from interaction with different matrix proteins. However, a fundamental unanswered question is how specific GTPase signaling pathways governing cell migration are regulated by different extracellular matrix proteins. Our hypothesis is that adhesion to different matrix molecules, such as collagen and fibronectin, will trigger differential regulation of guanine nucleotide exchange factors (GEFs) to regulate the cell migratory response. Although GEFs are known to activate the Rho GTPases, the possible regulation of their biochemical activity by cell interactions with different types of matrix is not known. Our recent studies have focused on identifying a matrix-specific GEF regulator and the mechanisms of its regulation of Rho family GTPases and their regulatory crosstalk in dictating the mode and speed of cell migration.